Bone is a complex and dynamic tissue composed of a mineralized connective tissue matrix and actively metabolizing cells whose principal role is the continuous remodeling of this matrix to permit repair and growth. These active cells include the osteoblast and osteoclast whose functions are the deposition and resorption of bone, respectively. Throughout life, a balance between the functions of these cells must be maintained in order that the integrity of the skeletal system is not compromised. A perfect example of the result of an imbalance in these processes is osteoporosis, a metabolic bone disease that is devastating in its personal and financial costs to society. In the past 25 years, much of the knowledge garnered about the biology and metabolism of the osteoblast has come from in vitro studies of cultured bone cells. A variety of factors (e.g. hormones, vitamins, growth factors, minerals, prostaglandins, substrata) are known to interact with bone cells and to influence their devel- opmental and metabolic behavior. Whereas the overall objective of this proposal is to further the understanding of bone cell metabolism, with particular reference to osteoporosis and fracture healing, the experiments described herein focus on the effects of vitamin C and/or a collagenous matrix on the expression of the osteoblast phenotype. Bone cells will be isolated from neonatal rat calvaria (a well-studied model) and placed into tissue culture under standard conditions. The principal experimental variables in these cultures will be the absence or presence of vitamin C and/or a collagenous matrix, and the subsequent effects on bone cell differentiation. The induction of alkaline phosphatase, an early indicator of the osteoblast phenotype, will be evaluated in addition to other markers of osteoblast development (e.g. osteocalcin production and vitamin D responses). The specific aims are to determine (1) which parameters (e.g. duration of exposure, effective concentration, cellular uptake, presence of other inducing agents) lead to the optimal effect of vitamin C on expression of alkaline phosphatase activity; (2) the nature of this induction (e.g. increase in activity per se or increase in enzyme protein (as measured by mRNA levels)); and (3) the role, if any, of cell proliferation and/or matrix synthesis and deposition on bone cell differentiation.